Objective: To investigate the effect of hypoxia inducible factor-1α (HIF-1α) activation on cholesterol homeostasis dysfunction in diabetic nephropathy (DN).
Methods: Rat models of type 1 diabetes established by intraperitoneal STZ injection were treated with intraperitoneal injection of Lificiguat (YC-1, a HIF-1α inhibitor). Human proximal tubular cell line HK-2 was incubated with cobalt chloride (CoCl2, 100 μmol/L) in the presence or absence of 30 mmol/L glucose for 24 h. Renal injury of the rats was assessed by measuring 24-h urinary total protein level and PAS staining of the renal tubules. Cholesterol deposition in rat kidneys and HK-2 cells were observed using a quantitative assay of total cholesterol and Filipin staining, and HIF-1α protein expression was detected using Western blotting, immunohistochemistry or immunofluorescence assay; the expressions of cholesterol metabolism-related proteins HMGCR, LDLr, CXCL16 and profibrogenic factors including TGF-β1 and CTGF were also analyzed.
Results: The diabetic rats showed significantly increased 24-h urinary protein level (P<0.001), obvious renal tubular injury, and increased renal cholesterol content (P<0.05) with significantly increased HIF-1α expression in the renal tubular (P<0.01). YC-1 treatment significantly ameliorated tubulointerstitial injury in the diabetic rats as shown by decreased 24-h urinary total protein (P<0.05) and reduced damage area of the tubules, and effectively decreased renal cholesterol levels and renal expression of HIF-1α (P<0.05). In HK-2 cells, CoCl2 stimulation in the presence of high glucose effectively activated HIF-1α expression (P<0.0001), aggravated cholesterol accumulation (P<0.05), and increased the expressions of HMGCR, LDLr, CXCL16, TGF-β1, and CTGF (P<0.05 or 0.01). Consistent with the in vitro study, YC-1 treatment also significantly decreased the expressions of cholesterol metabolism-related proteins and the profibrogenic factors in the renal tubules of the diabetic rats.
Conclusion: HIF-1α activation induces cholesterol homeostasis dysregulation possibly by upregulating the de novo synthesis and uptake of cholesterol, thereby aggravating tubulointerstitial injury in DN.
目的: 探讨糖尿病慢性缺氧时缺氧诱导因子-1α（HIF-1α）激活在糖尿病肾病（DN）胆固醇稳态失调中的作用及机制。
方法: 将SD大鼠随机分为3组，10只/组：对照组、糖尿病组（DM）、YC-1干预组（DM+YC-1）。DM和DM+YC-1组大鼠接受60 mg/kg链脲佐菌素单次腹腔注射构建糖尿病模型，对照组大鼠接受等量柠檬酸缓冲液腹腔注射。体外研究中，应用氯化钴（CoCl2，100 μmol/L）刺激有或无葡萄糖（30 mmol/L）条件下培养的人近端小管上皮细胞HK-2细胞24 h。采用24 h尿蛋白定量评估各组大鼠肾损伤情况，过碘酸-雪夫染色观察各组大鼠肾小管病理学损伤；总胆固醇定量检测及Filipin染色观察肾组织及HK-2细胞中胆固醇沉积情况；Western blotting、免疫组织化学染色检测大鼠肾组织HIF-1α的蛋白表达、细胞免疫荧光检测HK-2细胞中HIF-1α的表达情况，检测大鼠肾组织及HK-2细胞中胆固醇代谢相关分子3-羟基3-甲基戊二酰辅酶A还原酶（HMGCR）、低密度脂蛋白受体（LDLr）、CXC型趋化因子配体16（CXCL16）和纤维化相关分子转化生长因子-β1（TGF-β1）、结缔组织生长因子（CTGF）的蛋白表达。
结果: 与对照组相比，DM组大鼠24 h尿蛋白水平升高（P<0.001），肾小管损伤加重，且肾脏胆固醇含量升高（P<0.05）并伴有肾小管HIF-1α蛋白表达升高（P<0.01），而YC-1干预可降低糖尿病大鼠24 h尿蛋白水平（P<0.05）、明显减轻肾小管损伤、降低肾总胆固醇含量（P<0.05）。体外研究中，氯化钴有效诱导HIF-1α表达（P<0.0001）且显著增加HK-2细胞中的胆固醇含量（P<0.05），同时上调胆固醇代谢相关蛋白HMGCR、LDLr、CXCL16以及纤维化因子TGF-β1、CTGF的表达（P<0.01，P<0.05）。与细胞实验结果相一致，YC-1干预可下调糖尿病大鼠肾小管胆固醇代谢相关蛋白的表达，也减少纤维化因子TGF-β1（P<0.001）和CTGF（P<0.05）的表达。
结论: HIF-1α活化可通过增加胆固醇从头合成、摄取介导肾小管上皮细胞胆固醇稳态失调，加重细胞脂质沉积和损伤，促进DN进展。
Keywords: cholesterol; diabetic nephropathy; hypoxia inducible factor-1α; lipid accumulation; tubules.